Pmcs cypridina luc

We used secreted luciferase from Cypridina. The Pgk1 promoter (−648 to −50) was amplified from mouse genomic DNA by PCR and cloned into the Cypridina luciferase reporter plasmid pMCS-Cypridina Luc (Thermo Fisher Scientific) at the XhoI and BamHI restriction sites. sPLA₂-V-WT and sPLA₂-V KD cells as well as sPLA₂-V KD cells expressing sPLA₂-V-H48Q were transfected with Cypridina luciferase reporter vector using FuGENE HD Transfection Reagent (Promega) according to the manufacturer’s instructions. Renilla luciferase expression vector (pTK-Green Renilla Luc, Thermo Fisher Scientific) was co-transfected to serve as an internal control. At 24 hr post transfection, the culture medium and the cells were harvested. Cypridina luciferase activity in the harvested culture medium was measured using a SpectraMax L luminometer (Molecular Devices, San Jose, CA, USA) and a Pierce Cypridina Luciferase Glow Assay Kit (Thermo Fisher Scientific). Renilla luciferase activity in cellular lysates was measured on the luminometer using coelenterazine as a substrate. Cypridina luciferase activity was normalized to Renilla luciferase activity. Data are represented as fold-induction by normalizing the luciferase activity of the tested sample to that of the corresponding control sample.

SLC30A2 promoter‐Cypridina luciferase reporter plasmids, in which WT or mutant SLC30A2 promoter (from −363 to −32 with reference to the adenine base of the start codon methionine as +1) is inserted into the multiple cloning site of pMCS‐Cypridina Luc (Thermo Scientific, Waltham, MA, USA), were transiently transfected into A549, MCF7, or MDA‐MB‐231 cells, as described previously;²⁸ Renilla luciferase expression plasmid was simultaneously transfected. The activities of Cypridina and Renilla luciferase were measured using Pierce Cypridina Luciferase Glow Assay Kit (Thermo Fisher Scientific, Rockford, IL) and Renilla‐Glo Luciferase Assay System (Promega, Madison, WI, USA), respectively, using a Synergy H1 Hybrid multimode microplate reader (BioTek, Winooski, VT, USA). Cypridina luciferase activity was divided by that of Renilla luciferase for normalization of transfection efficiency.